Marcela Alexandra Coba Zapata - repositorio.usfq.edu.ec
Transcript of Marcela Alexandra Coba Zapata - repositorio.usfq.edu.ec
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UNIVERSIDAD SAN FRANCISCO DE QUITO USFQ
Colegio de Posgrados
CONTROL OF Salmonella enterica serovar Infantis
COLONIZATION IN POULTRY USING BACTERIOPHAGES
Marcela Alexandra Coba Zapata
Gabriel Trueba Piedrahita PhD
Director de Trabajo de Titulación
Trabajo de titulación de posgrado presentado como requisito
para la obtención del título de Master en Microbiología
Quito, 13 de marzo de 2020
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UNIVERSIDAD SAN FRANCISCO DE QUITO USFQ
COLEGIO DE POSGRADOS
HOJA DE APROBACIÓN DE TRABAJO DE TITULACIÓN
CONTROL OF Salmonella enterica serovar Infantis
COLONIZATION IN POULTRY USING BACTERIOPHAGES
Marcela Alexandra Coba Zapata
Firmas
Gabriel Trueba Piedrahita PhD
Director del Trabajo de Titulación
Gabriel Trueba Piedrahita PhD
Director del Programa de Maestría en
Microbiología
Stella de la Torre PhD
Decano del Colegio de Ciencias Biológicas
y Ambientales COCIBA
Hugo Burgos PhD
Decano del Colegio de Posgrados
Quito, 13 de marzo de 2020
© DERECHOS DE AUTOR
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MIEMBROS COMITÉ
Firmas
Gabriel Trueba Piedrahita PhD
Director del Trabajo de Titulación
Sonia Zapata PhD
Profesora del Programa de Maestría en
Microbiología
Patricio Rojas PhD
Profesor del Programa de Maestría en
Microbiología
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Por medio del presente documento certifico que he leído todas las Políticas
y Manuales de la Universidad San Francisco de Quito USFQ, incluyendo la Política
de Propiedad Intelectual USFQ, y estoy de acuerdo con su contenido, por lo que
los derechos de propiedad intelectual del presente trabajo quedan sujetos a lo
dispuesto en esas Políticas.
Asimismo, autorizo a la USFQ para que realice la digitalización y
publicación de este trabajo en el repositorio virtual, de conformidad a lo dispuesto
en el Art. 144 de la Ley Orgánica de Educación Superior.
Firma del estudiante:
Nombre: Marcela Coba Zapata
Código de estudiante: 00109091
C. I.: 1714850045
Lugar, Fecha Cumbayá, 13, marzo 2020
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Dedicatoria
A mis padres Carlota y Marcelo, a mi esposo Juan Pablo y a mis hijos Eva
Monserrat y Juan Emilio.
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Agradecimientos
Sinceros agradecimientos al Instituto de Microbiología de la USFQ, a la
USFQ y a PRONACA; al Dr. Alejandro Torres y al Dr. Gabriel Trueba por su
acompañamiento y dirección durante el trabajo de tesis, a la Dra. Sonia Zapata por
su asesoramiento y al Dr. Patricio Rojas por su acompañamiento en el proceso de
titulación, a las excelentes amigas que me dejó el programa MSc. Fernanda Loayza
y MSc. Lorena Mejía.
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Resumen
En Ecuador, la industria avícola es el principal proveedor de proteína
animal, aproximadamente 0.5 toneladas se consumen cada año y la industria crece
3%. Así como en otros países, Salmonella es un problema de salud pública
permanente asociado con esta industria. Nosotros describimos una estrategia
simple para controlar la colonización de Salmonella enterica serovar Infantis en
pollos de engorde utilizando bacteriófagos nativos administrados en el agua de
bebida. Los bacteriófagos fueron aislados desde efluentes, lavado de plumas de
aves y las camas en las instalaciones de producción avícola de una granja
ecuatoriana. Los fagos nativos aislados fueron evaluados cualitativamente en
cuanto a su capacidad de lisis específica de cepas de Salmonella enterica serovar
Infantis y amplificados en un coctel. La estabilidad del coctel fue demostrada en
soluciones de cloro en concentraciones de 0 a 4 ppm y en soluciones suplementadas
con un inhibidor de halógenos así como también con un protector viral. Se evaluó
la eficiencia del coctel suplementando el agua de bebida de un platel de producción
de pollos de engorde con 1000 aves y se comparó la frecuencia de aislamiento de
Salmonella Infantis en el lote tratado con un lote de igual tamaño en el cual no se
administró el coctel de fagos. No se detectó Salmonella enterica serovar Infantis
en los tamizajes de rutina de la producción de pollos de engorde donde los
bacteriófagos fueron aplicados. Por otro lado, en el grupo control, Salmonella
enterica serovar Infantis fue detectado con una frecuencia del 20% en los ciegos
de los pollos, 10% de las muestras de lavado de plumas, 33% de agua de
escaldadora y 20% de las muestras de agua del lavado final. En conclusión, el
aislamiento, amplificación y aplicación de cocteles de bacteriofagos nativos es una
herramienta útil en el biocontrol de la colonización de Salmonella enterica serovar
Infantis en la industria avícola.
Palabras clave: Salmonella enterica serovar Infantis, granjas avícolas,
bacteriófagos nativos, biocontrol
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Abstract
In Ecuador, the poultry industry is the main supplier of animal protein,
approximately 0.5 tons are consumed each year and the industry grows 3%. As in
other countries, Salmonella is a permanent public health problem associated with
this industry. We describe a simple strategy to control colonization of Salmonella
enterica serovar Infantis in broilers using native bacteriophages administered in
drinking water. The bacteriophages were modified from the effluents, the washing
of chicken feathers and the beds in the poultry production facilities of an
Ecuadorian farm. Specific native phages were qualitatively evaluated for their
specific lysis capacity of Salmonella enterica serovar Infantis strains and amplified
in a cocktail. The stability of the cocktail was demonstrated in chlorine solutions in
dimensions from 0 to 4 ppm and in solutions supplemented with a halogen inhibitor
as well as a viral protector. The efficiency of the cocktail was evaluated by
supplementing the drinking water from a broiler production plate with 1,000 birds
and the frequency of isolation of Salmonella Infantis was compared in the treated
batch with a batch of the same size in which the phage cocktail. Salmonella enterica
serovar Infantis was not detected in routine screenings of broiler production where
bacteriophages were applied. On the other hand, in the control group, Salmonella
enterica serovar Infantis was detected with a frequency of 20% in caecas, 10% of
the feathers washing samples, 33% of scalded water and 20% of the samples of
water from the final wash. In conclusion, the isolation, amplification and
application of native bacteriophage cocktails is a useful tool in the biocontrol of
colonization of Salmonella enterica serovar Infantis in the poultry industry.
Key Words/Index Terms: Salmonella enterica serovar Infantis, poultry
farms, bacteriophages, biocontrol
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Tabla de contenido
Resumen .................................................................................................................. 7
Abstract ................................................................................................................... 8
Introducción .......................................................................................................... 11
Métodología y diseño de la investigación ............................................................. 23
Resultados y discusión .......................................................................................... 28
Conclusiones ......................................................................................................... 30
Referencias ............................................................................................................ 30
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ÍNDICE DE TABLAS
Table 1. Human and animal diseases associated with host adapted or host
generalized serovars of Salmonella enterica subsp. enterica.
Table 2. Prevalence of Salmonella serovars associated to broiler
production in different countries
Table 3. Water requirement according to broiler growing phases. Viral
protector and phage cocktail supplementation in each phase
ÍNDICE DE FIGURAS
Figure 1. Doble layer analysis from diffent samples within chicken production
chain. There were documented those plates with abundant, meddle and less phage
plaques concentration and a negative sample to shown the diverse viral load found.
Salmonella enterica serovar Infantis were used as host bacteria.
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INTRODUCTION
Food borne diseases are an important concern in public health (Havelaar et
al., 2015; Torgerson et al., 2015). Death and diseases caused by contaminated food
are a constant threat worldwide (Torgerson et al., 2015). The WHO estimated that
the most frequent causes for food transmitted diseases are diarrheal disease agents,
especially norovirus, Campylobacter spp. and non-typhoid Salmonella enterica.
Other death causes are related to Salmonella Typhi, Taenia solium and Hepatitis A
virus (Torgerson et al., 2015). Salmonella is a common zoonotic and food born
pathogen and the third cause of human death associated to diarrheal diseases
worldwide (Ferrari, Rosario, Cunha-neto, Mano, & Figueiredo, 2019; Lei et al.,
2020).
Salmonella from commensal to intestinal pathogen
Salmonella is a cosmopolitan bacterium genus belonged to
Enterobacteracea family with high genetic diversity. Salmonella are non-spore
forming Gram negative bacilli with aerobic metabolism, H2S production and most
of them have peritrichous flagella. Salmonella comprises to species S. bongori and
S enterica, according White-Kauffmann scheme based on the surface antigens
expressed on lipopolysaccharide (LPS), flagella and capsular polysaccharide, there
are about 2,659 serovars of S. enterica (Ferrari et al., 2019). The most important
pathogens are classified among 1,547 serovars of Salmonella enterica subsp.
enterica, but less than 100 serovars are associated with human infections (Centers
for Disease Control and Prevention, 2020).
S. enterica colonizes the intestinal tract of almost every animal species wild,
domestic (pets) and farm ones. Also, Salmonella can survive under non favorable
environmental conditions, including desiccation and starvation (Raspoet, 2014) and
can cause contamination on poultry, swine and calve derived meat, which can occur
in any country, any place and any time (Ferrari et al., 2019).
Salmonellosis range from self-limiting gastroenteritis to severe bacteremia
and typhoid fever (S. E. Park, 2019; Tegegne, 2019). It depends on Salmonella
serovar and host interaction (Table 1.) (Lamas et al., 2018). There are adapted
serovars of Salmonella known as specialist because colonize and infect only a
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narrow range of host (Kingsley & Ba, 2000). This specialist includes those that
cause typhoid fever in humans (Table1). Moreover, there are specialist serovars for
animals too, such as Salmonella enterica subsp. enterica serovar Choleraesuis in
pigs, Gallinarum or Pollorum in poultry (Ferrari et al., 2019; Tanner & Kingsley,
2018). On the other hand, the generalist serovars are able to infect humans or
animals without restrictions, the most important examples are Enteriridis and
Thyphimurim serovars, which cause less severe symptoms that could be self-
limited with diarrhea and the main symptom (Ferrari et al., 2019).
Epidemiology and pathogenesis of salmonellosis
The infection dynamics of Salmonella depends on oral-fecal route and its
ability of colonize and infect their host. Around 52% of salmonellosis are non-
typhoidal and 37% are typhoidal Salmonella cases, which represents the 9% of
global diarrheal illnesses. However, 41% of all deaths associated with diarrheal
diseases are caused by Salmonella (Besser, 2018). It is also result of human
activities in farm animal industry, and food conservation process (Carrasco,
Morales, & García, 2012). Salmonellosis has a special attention for its social and
economic impact in productive schemes (Mouttotou, Ahmad, Kamran, &
Koutoulis, 2017; Sukumaran, Nannapaneni, Kiess, & Sharma, 2015). Fever,
abdominal pain and diarrhea are the most frequent symptoms of Salmonella sp.
infection. It is an emergent zoonosis with an annual worldwide incidence of 93.8
million people and with a cost that raised to 1000 USD for each case
(Evangelopoulou, Kritas, Christodoulopoulos, & Burriel, 2015).
The most common manifestation of Salmonella infections is gastroenteritis,
with a prevalence of 93.8 million cases worldwide each year (Vinueza, Cevallos,
Ron, Bertrand, & De Zutter, 2016). In European Union in 2008 there were 131,468
confirmed cases, representing the second cause of zoonotic diseases in humans
(Carrasco et al., 2012); in 2015 the United States 94,4625 salmonellosis cases were
reported resulting in 26 deaths (Lamas et al., 2018). In 2014, in Ecuador 3,373
cases were reported (Vinueza et al., 2016).
Virulence and host adaptation of Salmonella is due to virulence plasmids
(pSLT) and Salmonella pathogenicity islands (SPIs) (Fig. 3), which are evolutive
acquisitions(Lamas et al., 2018). There are five main SPIs (1–5). Proteins with
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invasive functions to epithelial cells are codified in SPI-1 and SPI-2 codifies
determinants for survival and replication inside host cells in Salmonella enterica
(Lamas et al., 2018; Tanner & Kingsley, 2018). A more efficient host colonization
by Salmonella enterica subsp. enterica is done by the posterior acquisition of SPI-
6, 2-aminoethylphosphonate metabolism, Island STM3779-STM3785, Island
STM4065-STM4080 and quorum sensing mechanism based on Autoinducer 2 (AI-
2) transport and processing that could be involved in communication with gut
microbiota, as virulence regulator and genetic exchange facilitator (Gast & Porter,
2020; Lamas et al., 2018; Tanner & Kingsley, 2018).
Pathogenesis model of Salmonella in humans began with the resistance of
Salmonella strains to stomach pH. Then Salmonella traverses the intestinal mucus
layer and adhere to intestinal epithelium by adhesins (codified in SP-3 and SP4).
Once attached, Salmonella express SPI-1 genes for the multi- protein complex
T3SS to be engulfed into the epithelial cell (Lamas et al., 2018). The cecal mucose
convert The H2S produced by the microbiota is used by cecal mucose cells to
produce thiosulphate as a protective response (S. E. Park, 2019). Neutrophiles use
thiosulphate and convert it in tetrathionate in the intestinal lumen. Salmonella uses
tetrathionate as respiratory electron acceptor and grows more than the fermenting
commensal bacteria. On the other hand, engulfed Salmonella located in Salmonella
Containing Vacuoles (SCV) express a second T3SS that lets bacteria survive and
replicate inside host cells (epithelial cells and macrophages) (Lamas et al., 2018).
Mature SCV migrate to the Golgi apparatus while Salmonella increase their
number by replication. Phagocytes and macrophages are also used for replication
inside SCVs when bacteria cross the epithelium and then phagocytes facilitate
bloodstream dissemination in the host (Lamas et al., 2018).
Salmonella in poultry industry
Food-animals, including pigs and poultry, could be colonized for different
Salmonella serovars (Aabo et al., 2002; Magwedere, Rauff, De Klerk, Keddy, &
Dziva, 2015). Salmonella Typhimurium, Enteritidis and Infantis are most
frequently associated with health problems in humans farm (Crim et al., 2014;
Hugas & Beloeil, 2014; Hungaro, Mendonça, Gouvêa, Vanetti, & Pinto, 2013; C.
J. Park & Andam, 2020). Colonized farm animals, without any observable clinic
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symptom are a big risk factor for food production industry (Fearnley, Raupach,
Lagala, & Cameron, 2011; Stevens, Humphrey, & Maskell, 2009).
Aviculture produce the major human´s source of protein from animal origin
(Shepon, Eshel, Noor, & Milo, 2016). In Ecuador, 30-32 Kg/year of chicken meat
are consumed per capita (Gutierrez, 2017). Around 46 million Gallus gallus were
reared in Ecuadorian Agricultural Production Units during 2012 (Data from
Continuous Agricultural Production and Production Survey. Ecuador). For 2017,
the annual production volume reached 250 million broilers (Gutierrez, 2017). The
Andean region produce the 68% of chicken and 85% are raised in commercial
poultry farms (Corporación Financiera Nacional Ecuador, 2016).
Among farm-animals, broilers production is challenged permanently by
Salmonella serovars colonization within chicken intestines or in their facilities
(Table 2). In Germany from 1991 and 1993, the prevalence of Salmonella in poultry
meat and its by-products the pathogen was present on 18% of the samples (Hartung,
1993), while in New Zealand, found it on 23/137 (17%) of non-frozen poultry and
2/17 (2%) of frozen poultry meat samples, (Rahman & Othman, 2017). But this
dynamic is not exclusive of geographic region or type of processing facilities.
Salmonella is a cosmopolitan bacterium and can cause contamination on poultry,
pork and beef, which can occur in any country, any place and any time. For the
European Union countries and 3 nonmembers, the general prevalence of
Salmonella was reported to be 3.37% within farms with rates varying from 0.08%
in Norway to 13.84% in Hungary in 2014 (Vinueza, 2017; Vinueza et al., 2016)..
In Brazil, 32% of carcases from 4 commercial farms were positive for Salmonella
(Fuzihara, Fernandes, & Franco, 2000) while United Kingdom found 8% positive
samples from analyzed carcasses in 2002. At the east of Azerbaijan, Dehnad, 2004,
examined 200 pieces of industrial and semi industrial processed chickens and found
31,5% positivity in the samples. Simmons et al, 2003, in the United States,
demonstrated 33.9% positivity for Salmonella on carcasses. Zeiton and Al-Edi,
2004, in Saudi Arabia, analyzed 360 frozen chickens and revealed the presence of
Salmonella on 20% of the samples. Other examples include Al Abidy, 2005, in Iraq
found 9,72% of frozen products positive for the bacteria. O the other hand, in
Sweden, where there is a small poultry industry, the prevalence of contaminated
poultry meat with Salmonella is low below 1%. In general, all this studies indicate
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that Salmonella contamination prevalence on poultry meat carcasses extended from
0.16% to 49% in the period from 1991 to 2006 (Rahman & Othman, 2017).
In Latin America, some Salmonella outbreaks in humans have been linked
to contaminated poultry consumption; nevertheless, data about Salmonella
prevalence in Latin America is scarce. Salmonella in poultry meat is associated
with fecal contamination from asymptomatic animals (Hugas & Beloeil, 2014).
Other sources are equipment in slaughterhouses, floor, or the manipulation of
asymptomatic workers. Final product could be contaminated with the pathogen in
any processing stage (Rahman & Othman, 2017). Different Salmonella serovars
have been detected on poultry final products which can cause disease in humans
(Antunes, Mourão, Campos, & Peixe, 2016).
In Pichincha, Ecuador, between 2013 and 2014, according to Vinueza, 15%
(n=388) of poultry commercial batches are Salmonella positive, besides mentions
that Venezuela has a similar incidence of 23% (n=332), unlike prevalence in Brazil
which is 5% (n=40) and in Colombia 65% (n=315).
Salmonella enterica serovar Infantis
In recent years, it has been noted a change in the prevalence of Salmonella
serovar Infantis in broilers raising systems as well as in human infection cases. The
reservoir of S. Infantis are farm animals in special poultry commercial farms
(Miller, Prager, Rabsch, Fehlhaber, & Voss, 2010). Its increased prevalence
worldwide is due to an evolutionary acquisition of a mega plasmid pESI that
confers bacteria antimicrobial and stress resistance, pathogenicity islands and
evolutive advantages to be a dominant serovar. Also confers a virulence factors but
with less expression level causing a non-severe symptoms when it infects human
hosts (Aviv et al., 2014).
The horizontally acquired mega plasmid pESI confers a clearly advantage
to this bacterium to become the most prevalent serovar isolated in poultry farm
screening (Aviv et al., 2014). Despite of S. Infantis don’t cause any disease in
poultry, but its presence in farms increases the probability of poultry carcasses
contamination and economical losses to the industry. In South Africa between 2013
and 2014, Salmonella Infantis was found among the most common 16 serovars
related to poultry farming (Magwedere et al., 2015). In the same way, countries
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like Cambodia, Vietnam and South Korea have S. Infantis as one of the
predominant serovars (Cui et al., 2016). Nowadays, Salmonella enterica subsp.
enterica serovar Infantis is one of the top ten serovars causing human salmonellosis
in both Europe and North America (Gymoese et al., 2019).In Ecuador, S. Infantis
is the most common serovar associated to poultry farms with a 83.9% prevalence
(Vinueza et al., 2016).
Detection of Salmonella requires technical expertise in microbiology and
an excellent technical performance due to several steps of enrichment and agar
culture of samples (Maddox, 2003). This difficulties and the need of rapid
responses and standardization drove the industry to develop more sensitive
diagnostics (Hendriksen, Wagenaar, Hendriksen, & Carrique-Mas, 2013).
Serotyping methods has been used since 1934 for description of endemic serovars
associated to animal colonization or animals or human infections. It consists in the
use of antisera specific for somatic or flagella antigens for an agglutination reaction
(Hendriksen et al., 2013). This method is wide used but it is expensive and focused
only in most prevalent serovars or those which presence alert the probabilities of
human disease associated with farm animals (Centers for Disease Control and
Prevention, 2020; Hendriksen et al., 2013).
There has been developed culture independent strategies to detect
Salmonella serovars. Commercial PCR-based methods like BAX®, ELISA- based
systems, Bioline Selecta, Bioline Optima and Vidas, or different strategies using a
non-commercial PCR system. The sensibility of methods range from 0.67 to 0.99
with VIDAS and ELISA based system with the poorest sensibility (Eriksson &
Aspan, 2007). More recently fluorogenic or real-time PCR methods have been
developed to generate quick results in Salmonella detection from different sources
using specific primers, Itsf and Itsr, for the internal transcribed spacer region of the
16S–23S rRNA gene (Cheung & Kam, 2012). Other isothermal amplification
methods under commercially protected protocols like 3M and ANRS are frequently
used in the industry (Bird et al., 2013; Foti et al., 2014).
Moreover, a rapid and precise typing system for Salmonella serovar
has been developed at the genetic level, commercial kits: Salm SeroGen (Salm
Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), xMAP (xMAP
Salmonella serotyping assay), and Salmonella geno-serotyping array (SGSA)
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(Yoshida et al., 2016). All these protocols need Salmonella colonies in pure culture
isolation. The identification of Salmonella serovars were correct in a range of 75%
to 100% of the nontyphoidal Salmonella samples. There were included serovars
Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow
(Yoshida et al., 2016). The molecular mechanism to S. Infantis detection consist
of targeted somatic and flagellar genes.
Control strategies of Salmonella colonization
Prophylactic antimicrobial administration has been used for elimination or
reduction of Salmonella sp. within normal microbiota of poultry (Evans &
Wegener, 2003). Other strategies are vaccines, prebiotics or probiotics under strict
quality control of facilities and workers (Antunes et al., 2016; Atterbury et al.,
2007; Chambers & Gong, 2011). However, none of them has achieved the
expected efficiency and the disease continues to be emergent (Laurimar Fiorentin,
Vieira, & Barioni, 2005). There are some research carried out with viruses that
infect bacteria ( bacteriophages), as an alternative, which has been promising in the
control of colonization, infection and spread of possible bacteria pathogen strains
in poultry (Atterbury et al., 2007; Laurimar Fiorentin et al., 2005; Grant, Hashem,
& Parveen, 2016; Spricigo, Bardina, Cortés, & Llagostera, 2013; Thung et al.,
2017; Yeh et al., 2017; Zinno, Devirgiliis, Ercolini, Ongeng, & Mauriello, 2014).
In the EU and western countries, poultry and derived products are the main
source of food infections caused by Salmonella, and from all the range of derived
products the main risk factors are uncooked eggs and poultry meat (Yeh et al.,
2017). To control of Salmonella in animals and animal products, several
alternatives have been proposed, (Spricigo et al., 2013). In the last decade, treating
human and animal diseases with antibiotics have become more difficult due to the
growing problem of bacterial resistance to antimicrobials (Busani et al., 2004), and
the risk to human health. The concern about antibiotic resistance has pushed the
market to seek alternative treatments. Among these intervention practices to reduce
or prevent the spreading of pathogens are the use of different animal genetic lines
that control the immune response, chemical treatments to avoid the vertical
transmission of Salmonella by immersion in oxygen peroxide and phenol of the
recent harvested eggs reducing its bacterial load (Doyle & Erickson, 2004), general
guides of sanitization by using chemicals, temperatures, pressure for a correct
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cleaning and disinfection of all areas. Another alternative practice of raising
poultry selecting different bed materials, treatment with different chemicals or
composting processes, water sanitization, food supplements with prebiotics,
application of probiotics for bacterial competitive exclusion, additives to improve
immune responses, vaccines and bacteriophages (Doyle & Erickson, 2004).
Bacteriophages.
Bacteriophages are viruses widely spread in nature, are ubiquitous entities
and can be found in sea, soil, deep sea vents, and gastrointestinal tract of humans
and animals (Belay, Sisay, & Wolde, 2018). Phage life cycle is strictly associated
with the bacterial cell, they have been denominated as molecular parasites because
they lack of cell structures and enzymatic systems necessary for food absorption,
protein synthesis or new particles construction, and as incomplete organisms they
can replicate only inside a living cell (Wernicki, Nowaczek, & Urban-chmiel,
2017).
Bacteriophages were discovered by Twort (1915) as “unidentified
molecules which inhibit bacterial growth”, but in 1917 D’Herelle was the first one
to isolate and characterize phages (Brown, Lengeling, & Wang, 2017; Kutter et al.,
2010). The International Committee on Taxonomy of Viruses, EC 48, Budapest,
Hungary, August 2016 (ICTV) imposed some criterion of taxonomy based on
genome type and virion morphology (Wernicki et al., 2017). Moreover, the use of
proteomics helps to classify viruses in 873 species, 204 genera and 14 subfamilies
(Adriaenssens & Brister, 2017). Other factors like host preference, auxiliary
structures such as tails or envelopes are considered (Orlova, 2012). Based on
nucleic acids, phages can be divided in three groups, double helix DNA, single
chain DNA and RNA; most phages described have double helix DNA genome.
Other important feature is capsid symmetry, differentiating two groups isometric
(polyhedral) and helicoidal (spiral) (Wernicki et al., 2017). It has been proposed
that phages are the most abundant form of life in the planet, by 2017 more than
25,000 sequences of nucleotides have been saved on databases, and this abundance
of phages in nature is what it makes so great when investigated as they are easily
found (Haq, Chaudhry, Akhtar, Andleeb, & Qadri, 2012)
According to the type of infection, phages can be divided in two groups:
hose that cause a lytic infection and the other that cause lysogenic, or temperate,
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type of infection (Orlova, 2012). The replication of phages is like viruses that infect
eukaryotic cells: starts with adsorption, penetration, nucleic acids replication,
phage formation and its release from de host cell (Kutter et al., 2010; Nelson, 2004;
Wernicki et al., 2017). During a bacterium infection by lytic phages, DNA is
released and induces switching of the protein machinery of the host bacterium.
Whit this change, 50-200 of new viral particles could be produced causing the death
of host bacterium (Orlova, 2012). On the other hand, lysogenic infection is
characterized by integration of the phage DNA into the host cell genome, so that it
could be replicated and vertically transmitted to new bacteria cells as a prophage
(Orlova, 2012; Wernicki et al., 2017) This phase could be reverted under stressful
conditions surrounding bacteria population and a lysogenic phage could be a lytic
one (Wernicki et al., 2017).
Analysis of phages with lysogenic or lytic mode of infection has shown that
there is a tremendous variety of bacteriophages. Some phages show specific affinity
with unique types of bacteria, while others show affinity for a wider group, the
specifics of this affinity is determined by the presence of receptors in the surface of
bacterial cells, as LPS fragments, fimbria and other surface proteins. Under lytic
phages activity the adhesion process to the bacterial cell consist on the union
between phages protein and the cell receptors like teichoic acid and lipoteichoic for
Gram positive or LPS for Gram-negatives (Wernicki et al., 2017). Then it has a
phase of penetration into the genetic material and it reaches the eclipse, where the
replication of nucleic acid and the proteins part of the capsid structure occurs, while
genetic material replication of the bacteria is on hold, then the phage is formed and
mature followed by the release of the newly formed phages producing bacterial
lysis. Phages known for lytic activity are T1 and T4. On the other hand, under
lysogenic cycle, after forming a prophage, the phage cycle is blocked and enters a
latency period which can be interrupted by external factors as sun light, UV
radiation or antibiotics. Examples from phages with lysogenic activity include
MM1 and 11 phages (Kutter et al., 2010; Nelson, 2004; Wernicki et al.,
2017).
Salmonella is an Enterobacteriacea that could be easily affected by specific
phages. S. Thiphymurium for example is the target bacteria for P22 bacteriophage,
which belongs to Podoviridae family. P22 uses a non-contractile tail to adsorb to
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Salmonella surface. Phages L, MG178 y MG40 have Salmonella Thiphymurium as
target (Labrie, Samson, & Moineau, 2010). Other Salmonella serovars could be
host for Epsilon 15 bacteriophage (Orlova, 2012). In nature, phages and bacteria
are in continuous cycles of co-evolution (Chaturongakul & Ounjai, 2014). Thus,
bacteria resistance to phage infection can occur and are studied under controlled
laboratory conditions, with a unique bacterial host-phage model. However, at
environmental conditions a conjunction of phage resistance mechanism could be
working at the same time (Labrie et al., 2010). Mechanisms used by bacteria for
phage resistance are listed from an interesting review done by (Kurtböke, 2012):
- Phage adsorption inhibition
o Blocking phage receptors: Changes on three-dimensional
conformation of surface structures or their adaptation
o Extracellular matrix production confers protection by a physical
barrier, phage cannot interact with surface molecules
o Competitive inhibitor production, that mean environmental
molecules that are in the bacteria niche and can interact with
phages receptors
- Blocking phage DNA entry
o Proteins from superinfection exclusion system consist of
membrane anchored or associated with membrane components
causing an inhibition of DNA injection into cells, the transfer of
viral DNA into bacterial cytoplasm or by inhibition of phage
lysozyme.
- Cutting phage nucleic acids
o Restriction – modification system classified from type I – type
IV groups. This system consists of bacterial enzymes that
recognize and degrade non-methylated DNA by restriction
enzymes and depend on the restriction methylase enzymes ratio
and the number of restriction sites in virion genome.
o CRISPR-Cas system is acronym of clustered regularly
interspaced short palindromic repeats ant its association with cas
proteins. The CRISPR loci are composed of 21–48 bp direct
repeats interspaced by non-repetitive spacers (26–72 bp) and
21
flanked by cas genes. When a virus infects a bacterium, one new
repeat-spacer unit at the 5′ end of the repeat-spacer region of a
CRISPR locus is acquired. This proto spacer sequence is
identical to that found in the viral genome and it is used to
identify and degrade incoming viral or plamid DNA.
- Abortive infection systems (Abi)
o RexA-RexB system: Blocks the replication, transcription or
translation of phage through a two-component system (RexA-
RexB), that is activated by the viral DNA-protein complex.
RexA is an intracellular sensor that forms and homoduplex and
activates RexB wich is membrane anchored ion channel. The
activation of RexB drives a drop in the cellular ATP level by
reduction on membrane potential. In consequence there are
decrease in macromolecules synthesis and stops phage infection
(Labrie et al., 2010)
o Lit-PrrC system that inhibits the phage translation and probably
activates IC R–M system. Activated PrrC cleaves tRNA block
phage infection in consequence.
o Others that involve resistance-induced physiological changes
that could destroy bacteria cell too.
The ubiquity of bacteriophages in any environment facilitates their isolation
and description of their suitability to be use against bacteria pathogens in human or
animal infections or as colonizers (Brown et al., 2017; Chaturongakul & Ounjai,
2014). The specificity of isolated phage limits its effectiveness when it is compared
with antimicrobial administration (Brown et al., 2017), but due to the raising
antimicrobial resistance phenotype of interest bacteria phage therapies could be an
extraordinary tool in eliminating bacterial infections (Belay et al., 2018). In this
context, poultry industry has been used bacteriophages to treat animals’ diseases
(Belay et al., 2018; Wernicki et al., 2017) whcih improves animal productivity and
health. Salmonella Infantis in poultry industry is a selected target because its high
prevalence (Miller et al., 2010). The emergence of new dominant S. Infants linage
with mega plasmid, potential spread to humans and the fast antimicrobial resistance
determinants acquisition is a high relevant challenge. Many publications have
22
showed the biological control of resistant bacteria with bacteriophage cocktail
applications (Belay et al., 2018; Borie et al., 2008; Rahaman et al., 2014; Zhang et
al., 2010). However, commercial products containing phages could be less
effective along time vs. native phages cocktails periodically isolated. The aim of
this study was to isolate native bacteriophages from a poultry farm samples
including sewage, effluents, bedding, and feathers and evaluate their specific
activity against Salmonella enterica serovar Infantis as a potential biocontrol tool
its colonization in poultry. The bacteriophages have had a special interest and their
main applications are: alternative to antibiotics against bacterial pathogens
including food pathogens (phage therapy); screening tools based on phage-display;
or genetic tools for pathogenic bacteria detection (phage-typing) (Belay et al.,
2018). As biologic control agent they had great success rates due to its capacity to
infect a wide range of bacterial species, a serotype or a strain. Increasing bacterial
resistance to antibiotics and antibiotic use restrictions, create the need of new
alternatives such as the isolation of native bacteriophages (Wernicki, Nowaczek, &
Urban-chmiel, 2017).
For bacteriophage isolation, the host strains must be those serovars with
major prevalence in animal farms to control their own colonization and reduce their
prevalence withing the broiler complex., therefore we used S. Infantis for phage
enrichment culture due to the high relevance of this serovar as the main concern in
Ecuadorian poultry industry (Vinueza et al., 2016) as well as other authors used
different Salmonella serovar including those with some association with human
disease like Salmonella Enteritidis, Salmonella Infantis, Salmonella Heidelberg,
and Salmonella Typhimurium (Rivera et al., 2018). Other authors reported the use
of different strains of Salmonella as hosts for bacteriophage isolation without
restriction of wide range of lytic activity of bacteriophages but focused in isolation
from bacteria of public health interest (Petsong, Benjakul, Chaturongakul, Switt, &
Vongkamjan, 2019; Phothaworn et al., 2019). Multi-strain Salmonella enrichment
worked in the bacteriophage isolation as was shown by (Petsong et al., 2019),
therefore, it could be an important consideration for future perspectives focus on
wide range host bacteriophage isolation. In this study we tested the application of
native bacteriophages (using S. Infantis host) in drinking water to reduce or
eliminate S. Infantis in chicken carcasses.
23
METHODS AND STUDY DESING
This study was approved by the animal health committee and respect the
technical and ethical working conditions of animal welfare in the industry,
according with universal rules for animal use in experimental proposes. The farm
included for this study belonged to an integrated broilers batch followed for the
entire production and processing cycle (One day age broiler reception, feed and
growing, slaughtering, carcasses processing and marketing) at poultry farms and
slaughter facilities in Santo Domingo de los Tsachilas - Ecuador. Three phases were
performed during the study: 1) Phage isolation from environmental and chicken
samples; 2) phage sensibility and specificity and 3) intervention with a viral
cocktail. All broilers used had the same age and were housed on the same day.
1) Phage isolation from environmental and chicken samples
Bacteria strains.
For bacteriophage isolation, we used two strains of S. Infantis (identified as
U and P strains) were used to select bacteriophages. The bacterial species were
confirmed by serologic test with a ready to use rabbit antiserum (SSI Diagnostica
A/S, Denmark) and molecular techniques (Kim et al., 2006). Additionally, The
Microbiology Institute of San Francisco University donated strains of Salmonella
enterica serovars Typhimurium, S. Enteriditis and Eschericia coli. For sensitivity
test we used 44 S. Infantis strains from the bacteria collection kept in the diagnostics
laboratory of the industry. We focused on S. Infantis, for bacteriophage isolation
under company demands for the high prevalence of this strain in Ecuadorian
poultry farms (Vinueza et al., 2016).
Phage isolation.
We used tryptic soy broth, tryptic soy with 0,7% of agar and tryptic soy agar
(1.5% of agar) for enrichment and isolation with doble layer method as an
economical and technically efficient protocol for phage isolation (Rivera et al.,
2018). Previous nonpublished data from de industry let us know that the most
efficient place to isolate phages are broiler houses and slaughtering facilities. With
completely random sampling design, we selected 5 samples from scalding water
from chickens, 5 samples from chicken feathers water, 5 samples from water used
24
to flush chicken carcasses and 10 samples from chicken bed, moreover 5 samples
of turkey carcasses flush water and 5 samples from scalding water from turkeys
were collected. Samples were transported in ice to the laboratory facilities withing
2 hour after collection. Twenty-five milliliters of each liquid sample or 25 g of
chicken bed were placed in a flask with 225mL of Buffered Peptone Water (BPW).
For native bacteriophages isolation, a target bacteria S. Infantis culture was
prepared 18h before the assay. Briefly, 0.2mL of S. Infantis culture and 2mL of
each liquid sample prepared with BPW were added to 5mL of Tryptic Soy Broth.
These tubes were incubated at 37°C by 24h. Then, each sample was centrifuged at
15,652 g for 10 minutes. One milliliter of each supernatant was dispensed in a
1,5mL sterile tube and 0.2mL of chloroform was added. These bacteriophage
cocktail samples (BCS) were stored at -20°C for further analysis. Presence of S.
Infantis enriched bacteriophages were evaluated as following: tubes with molten
semisolid (0.7% agar) tryptic soy culture media at 45 °C were mixed with 0.3mL
of 18 hours culture of S. Infantis and 0.2mL of the BCS. After mixing, the semisolid
medium was dispensed in petri dish containing a solidified layer of tryptic soy agar.
Petri dishes were incubated at 37°C for 18 hours. Clear zones (plaques) in the
bacterial lawn indicated the presence of lytic phages (Kropinski, Mazzocco,
Waddell, Lingohr, & Johnson, 2009). Plaques were collected using a cut sterile
plastic pipette tip and suspended in 1mL of salt magnesium (SM) buffer and stored
at -20°C (Clokie & Kropinski, 2009). These phage lysates (PL) were used in further
assays to test the host specificity and to amplify a phage cocktail. The strong lysis
ability was the characteristic that we use to select phages in our cocktail (Petsong
et al., 2019). These features were valued in front to the halo diameter, de abundance
al transparency of the plaques (Rivera et al., 2018).
2) Isolated phage sensibility and specificity testing
Bacterium host specificity of native phage lysates.
Phage lysates were tested against S. Infantis (P and U), Escherichia coli, S.
enterica serovar Typhimurium and S. enterica serovar Enteritidis and 44 S. Infantis
strains from a collection kept in diagnostic laboratory of the industry. One strain
was isolated from chickens’ bed, 10 strains were isolated from chicken meat and
the other were isolated from environmental samples. A 24h- bacterial culture was
25
adjusted to 0.5 McFarland scale using sterile TSB. Then, 100μL of this bacteria
dilution were inoculated in 5mL of molten TSA (0.7% agar) and overlaid onto a
cell of tri Petri dish. Inoculated culture media plates could solidify for 15 min and
were incubated at 37°C for 18h. Each native PL obtained was spotted onto lawns
of a host bacterium strain culture using a sterile inoculating loops with 2mm of
diameter. Plates were incubated at 37°C for 18h and the appearance of clear zones
of lysis were describes as positive bacteriolytic activity (Gencay & Brondsted,
2019). Lytic capability was tested with double layer agar plate using S. Infantis
isolate as target (Kropinski et al., 2009). Four phage lysates with the best lytic
activity (higher clear zone diameter) and greater spectrum against S. Infantis were
selected to be used in a cocktail.
3) Selection of phage cocktail amplification and administration in
water source.
Cocktail preparation in base of selected phage lysates amplification.
To scale up the four selected phage lysates, each one was amplified as
follow: 1mL of PL was inoculated in a fresh S. Infantis culture, in a final volume
of 250mL of TSB. After overnight incubation at 37°C, the culture was filtered with
a vacuum system using filter cops (FILTERFLOCKEN MAN 201) which trapped
biomass and then 0.2 μm diameter pores filters were used to guaranty any bacteria
contamination in the final filtrate solution. Control of non-bacteria contamination
were done in nutrient culture media and XLT4 media as selective one. Filtrates
were stored under freezing conditions at -20 ° C.
Phages cocktail stability in vitro test on water system.
In broilers house, drinking water was treated with chloride solutions to
avoid probable pathogens present in water. Then, a commercial halogen neutralizer
BALMAR® or a viral protector PROVIR ® was added to water. We tested the lytic
ability of phage cocktail in water supplemented with chloride solution at 0, 1 2 3
and 4 ppm as final concentration, then commercial halogen neutralizer, were added
following manufacture instructions with a final concentration of 1Kg in 100L of
water. A similar batch of chloride water was used to be supplemented with the viral
protector under the same conditions and following the manufacture
recommendations. An experimental unit was defined as a flask with 500mL of
26
chloride water (0, 1 2 3 and 4 ppm) treated with the halogen neutralizer or the viral
protector and with an inoculum of prepared phage cocktail in a ratio of 1:100. Three
repetition of each experimental unit were performed. Chlorinated water at the same
concentrations was used as control. The commercial solution used was a factor in
this experimental design, the other factor was the time of phage cocktail exposure
at 0, 1, 2, 3, and 4 hours.
To evaluate the stability of phage lytic activity, a doble layer assay were
performed for each experimental unit. Briefly, 0.2mL of 18h S. Infantis culture and
0.1mL of each liquid sample were added to 5mL of molted Tryptic Soy Broth with
0,7% of agar. These mixes were poured on previously prepared tryptic soy agar
plates. After 24h incubation at 37°C, the plaques were analyzed.
Bacteriophage cocktail application in drinking water system.
Under farm conditions, drinking water is treated with 1ppm of chloride
solution. Previous nonpublished data were used to calculate the water requirement
in each growing phase of broilers described in the table 3. Additionally, the
continuous surveillance program for Salmonella detection in farms environment
and the whole poultry productive chain let us know the prevalence of Salmonella
in different stages. Based in those nonpublished data, we selected two Salmonella
Infantis positive broiler sheds with 1,000 animals each and we follow them during
their whole life until their slaughter process under the strictly protocols of animal
welfare and slaughter in chicken production industry.
The experimental design consisted of 1,000 of chickens selected as control
group and 1,000 chickens used as treatment group in a different barn. Both were
separately managed with the same light and feed programs, with a density of 10
chickens/m2 area. Bacteriophage cocktail suspension was applied 1:100 in the
treated water after a 2-hour water starvation for chicken. The viral protector was
colored in blue to confirm all chickens take water and the phage cocktail doses
included in. Phage cocktail suspension was applied every 5 days for 4 times
increasing the total water volume administered according to the chicken age. No
screening or control samples were taken either chickens nor barn environment
during chickens’ life to avoid stressors or non-controlled factors that could affect
the chicken’s health.
Salmonella Infantis screening in slaughtering facility.
27
The experimental group (treatment A) was slaughtered at the first turn in
the morning and the control group (treatment B) was slaughtered the next day to
avoid any cross contamination. Liver, caeca, feathers, scalding water, carcass rinse
water and finished product samples. Samples were pre-enriched in BPW during 18
hours in 37°C. The screening for Salmonella sp. was done following ANSR
(Apracom S.A.) protocols (Foti et al., 2014). Positive samples were analyzed to
confirm the detection and identification of Salmonella Infantis following standard
microbiology procedures with XLT4 y BPLS culture media and serology test
28
RESULTS AND DISCUSSION
Our results indicated that one application of bacteriophage cocktail was
effective in the reduction of Salmonella in the digestive tract of poultry which is in
agreement with previous reports (L Fiorentin, Vieira, Barioni Júnior, & Barros,
2004). The results showed a complete absence of S. Infantis in all samples taken
from chickens in the treatment group. Meanwhile, in the control group 20% of
caeca, 10% of feathers, 33% of scalding water and 20% of carcass rinse water were
positive for S. Infantis detection. In other studies, periodic application of a
combination between a phage cocktails and probiotics mixture, applied orally on
poultry showed 10 times less presence of studied Salmonella serovars in the liver,
spleen, caeca’s and ileum than untreated poultry (Laurimar Fiorentin et al., 2005).
Also, Filho et al. showed that the oral administration of a phage cocktail prevented
S. Enteriditis colonization for short period of time (48 hours). In our study, the
presence of S. Infantis were evaluated after 42 days of production period, taking
samples from liver, caeca, feathers, scalding water, carcass rinse water and finished
product.
To isolate bacteriophages, we used samples from scalding water (n = 5),
chicken feather water (n = 5), flush chicken carcasses water (n = 5) and chicken
bed (n = 10). We found that 88% of the samples yielded bacteriophages (31 of 34)
that was lower than the reported for Chilean broiler farms in which 97% of
processed farm samples were positive for bacteriophages (Rivera et al., 2018).
To choose bacteriophage candidates for biocontrol applications, the transparency
of litic halos has been taken as qualitative variable (Rivera, et al, 2018). In this
study all bacteriophage isolates showed clear plaques with the same or similar
transparency. Those mean that all bacteriophage isolates had the same potential to
be part of the final control by their lytic activity against Salmonella Infantis. To
evaluate the sensibility of isolated bacteriophage activity, we used 44 Salmonella
Infantis strains from our own collection. We did this assay because we wanted to
evaluate if the permanent changes on adsorption molecules in bacteria or the
development of phage resistance mechanisms could affect the affinity of the native
bacteriophages against Salmonella Infantis strains (n=44) isolated from different
point times in the industry (Wernicki et al, 2017, Kurtböke et al, 2012). In our
29
study, 100% of isolated phages (n=30) showed clear plaques on the primary
cultures (Figure 1). The plate lysates were evaluated against different strains of S.
Infantis, 27 out of 44 strains were sensitive to all phages; 6 were sensitive to a
cocktail containing a combination of 2 to 29 different phages and 11 strains were
resistant to all phages and probably it explains that 25% of Salmonella Infantis
strain collection were resistant to all phages lysates obtained, highlighting that
Salmonella strains and phage lysate were isolated in different settings within the
industry and the difference among used Salmonella strains is only the time of
isolation. In Rivera et al study, they reported a narrow range of lytic activity of
isolated bacteriophage in 9 phage isolates, but they worked with three Salmonella
serovar strains as target bacteria. We obtained 27 phages with narrow host
specificity affecting only S. Infantis. The presence of S. Infantis lytic phages in high
frequency suggest that the environmental presence of this strain along the chicken
production chain, since the raising house to slaughtering, but also means that the
more dangerous non typhoid pathogen strains like S. Thiphymurium and S.
Enteritidis are not present in the same prevalence (Higgins et al., 2008).
30
CONCLUSIONS
We demonstrated that the use of native bacteriophages is an efficacious procedure
to reduce S. Infantis in samples from broilers or environment associated with
chicken industry. Successful control of Salmonella serovars and other pathogens
associated with food animal farms have been shown previously. It is necessary to
conduct additional studies to determine the cocktail efficacy overtime. It is known
that bacteria acquire immunity to phages, and we assessed that the best and cheaper
strategy against this co-evolution is the periodic isolation of new phage collection.
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Table 1. Human and animal diseases associated with host adapted or host generalized serovars of Salmonella enterica subsp. enterica
Serovars Host range
classification
Natural hosts Disease Symptoms or sign(s) Rare hosts
S. Typhi Host restricted Humans Typhoid fever Septicemia, fever -
S. Paratyphi A Host restricted Humans Paratyphoid fever Septicemia, fever -
S. Paratyphi B Host restricted Humans Paratyphoid fever -
S. Paratyphi C Host restricted Humans Paratyphoid fever -
S. Typhimurium Host generalised Humans Gastroenteritidis Diarrhea, dysentery, fever None
Bovines Salmonellosis Diarrhea, dysentery, septicemia, fever
Swine Salmonellosis Diarrhea
Sheep Salmonellosis Diarrhea, dysentery, septicemia
Horses Salmonellosis Septicemia, Diarrhea
Rodents Murine typhoid Septicemia, fever
Poultry
S. Enteritidis Host generalised Humans
Gastroenteritidis Diarrhea, dysentery, fever Swine and
bovines
Rodents Murine typhoid Septicemia, fever
Poultry
S. Infantis Host generalised Human Gastroenteritidis Diarrhea, dysentery, fever
Poultry
S. Dublin Host adapted Bovines Salmonellosis Diarrhea, dysentery, septicemia, fever abortion Humans
S. Choleraesuis Host adapted Swine Pig paratyphoid Skin discoloration, septicemia, fever Humans
S. Gallinarum Host restricted Poultry Fowl typhoid Diarrhea, comb discoloration, septicemia None S. Pullorum Host restricted Poultry Pullorum disease Diarrhea, septicemia
S. Typhisuis Host restricted Swine Chronic paratyphoid Intermittent diarrhea S. Abortusovis Host restricted Sheep Salmonellosis Septicemia, abortion, vaginal discharge Diarrhea,
dysentery
S, Abortusequi Host restricted Horses Salmonellosis Septicemia, abortion, Diarrhea
2
Table 2. Prevalence of Salmonella serovars associated to broiler production in different
countries
Country/Region Serovar Year % Source
European Union
S. Infantis 2011-2013 26,5% (Atunnes, 2015) (Vinueza-Burgos et al., 2016)
S. Infantis
2014
43,4%
(Vinueza-Burgos et al., 2016) S. Mbandaka 13,5%
S. Livingstone 7,3%
S. Enteriditis 7,3%
Japan S. Infantis 2004-2005 (Assai, 2006)
United States
S. Enteritidis
2016
16,8%
(CDC Report, 2016)
S. Newport 10,1%
S. Typhimurium 9,8%
S. Javiana 5,8%
S. I4(5),12:i:- 4,7%
S. Infantis 2,7%
Venezuela S. Parathyphi B
(Vinueza-Burgos et al., 2016) S. Heidelberg
Colombia
S. Paratyphi B Dt +
(Vinueza-Burgos et al., 2016) S. Heidelberg
S. Enteriditis
S. Typhimurium
Peru
S. Infantis 84,0%
(Vinueza-Burgos et al., 2016)
S. Enteritidis 5,0%
S. Senftenberg 6,0%
S. Debry 1,7%
S. Kentucky 1,7%
Brasil
S. Enteritidis
(Vinueza-Burgos et al., 2016) S. Infantis
S. Typhimurium
S. Heidelberg (37)
Pichincha - Ecuador
S. Infantis
2013-2014
83,9%
(Vinueza-Burgos et al., 2016) S. Enteriditis 14,5%
S. Corvallis 1,6%
3
Table 3. Water requirement according to broiler growing phases. Viral protector and
phage cocktail supplementation in each phase
Application Broiler age
(Days) Broiler
(n) Water
volume (L) Phage cocktail
volumen (L) Coloured solution of
viral protector (L)
1 20 1,000 60 0.600 0.600
2 25 1,000 60 0.600 0.600
3 30 1,000 80 0.800 0.800
4 35 1,000 100 1,000 1,000
Figure 1. Doble layer analysis from diffent samples within chicken production chain.